The present invention is directed to novel pyrazolobenzodiazepines which inhibit cyclin-dependent kinases (CDKs), in particular CDK2. These compounds and their pharmaceutically acceptable salts, and prodrugs of said compounds, are anti-proliferative agents useful in the treatment or control of cell proliferative disorders, in particular cancer. The invention is also directed to pharmaceutical compositions containing such compounds, and to methods for the treatment and/or prevention of cancer, particularly in the treatment or control of solid tumors. The compounds of the invention are especially useful in the treatment or control of breast, colon, lung and prostate tumors. The invention is also directed to intermediates useful in the preparation of the above anti-proliferative agents.
Uncontrolled cell proliferation is the hallmark of cancer. Cancerous tumor cells typically have some form of damage to the genes that directly or indirectly regulate the cell-division cycle.
Cyclin-dependent kinases (CDKs) are enzymes which are critical to cell cycle control. See, e.g., Coleman et al., xe2x80x9cChemical Inhibitors of Cyclin-dependent Kinases,xe2x80x9d Annual Reports in Medicinal Chemistry, vol. 32, 1997, pp. 171-179. These enzymes regulate the transitions between the different phases of the cell cycle, such as the progression from the G1 phase to the S phase (the period of active DNA synthesis), or the progression from the G2 phase to the M phase, in which active mitosis and cell-division occurs. See, e.g., the articles on this subject appearing in Science, vol. 274, Dec. 6, 1996, pp 1643-1677.
CDKs are composed of a catalytic CDK subunit and a regulatory cyclin subunit. The cyclin subunit is the key regulator of CDK activity, with each CDK interacting with a specific subset of cyclins: e.g. cyclin A (CDK1, CDK 2). The different kinase/cyclin pairs regulate progression through specific stages of the cell cycle. See, e.g., Coleman, supra.
Aberrations in the cell cycle control system have been implicated in the uncontrolled growth of cancerous cells. See, e.g., Kamb, xe2x80x9cCell-Cycle Regulators and Cancer,xe2x80x9d Trends in Genetics, vol.11, 1995, pp.136-140; and Coleman, supra. In addition, changes in the expression of or in the genes encoding CDK""s or their regulators have been observed in a number of tumors. See, e.g., Webster, xe2x80x9cThe Therapeutic Potential of Targeting the Cell Cycle,xe2x80x9d Exp. Opin. Invest Drugs, Vol. 7, pp. 865-887 (1998), and references cited therein. Thus, there is an extensive body of literature validating the use of compounds inhibiting CDKs as anti-proliferative therapeutic agents. See, e.g. U.S. Pat. No. 5,621,082 to Xiong et al; EP 0 666 270 A2; WO 97/16447; and the references cited in Coleman, supra, in particular reference no. 10. Thus, it is desirable to identify chemical inhibitors of CDK kinase activity.
It is particularly desirable to identify small molecule compounds that may be readily synthesized and are effective in inhibiting one or more CDKs or CDK/cyclin complexes, for treating one or more types of tumors.
Several classes of compounds that inhibit cyclin-dependent kinases have been and are being investigated as therapeutic agents. These are, for example, as follows:
Analogs of Flavopiridol:
U.S. Pat. No. 5,733,920 (Mitotix)
WO 98/1344 (Bristol-Myers Squibb)
WO 97/42949 (Bristol-Meyers Squibb)
Purine Derivatives:
WO 98/05335 (CV Therapeutics)
WO 97/20842 (CNRS)
Acridones and Benzothiadiazines:
WO 98/49146 A2 (US Dept. of Health and Human Services)
Antisense
U.S. Pat. No. 5,821,234 (Stanford University).
Furthermore, certain N,N-substituted dihydropyrazolobenzodiazepines have been disclosed in an article discussing CNS-acting compounds. See, M. A. Berghot, Arch. Pharm. 325:285-289 (1992).
There continues to be a need for easily synthesized, small molecule compounds for the treatment of one or more types of tumors, in particular through regulation of CDKs. It is thus an object of this invention to provide such compounds and compositions containing such compounds.
The present invention relates to pyrazolobenzodiazepines capable of inhibiting the activity of one or more CDKs, in particular CDK2. Such compounds are useful for the treatment of cancer, in particular solid tumors. In particular the compounds of the present invention are especially useful in the treatment or control of breast, colon, lung and prostate tumors. The invention is also directed to intermediate compounds useful in the preparation of the above-mentioned pyrazolobenzodiazepines.
The compounds of the present invention are compounds of formula I below 
and prodrugs and metabolites of the foregoing compounds, as well as pharmaceutically acceptable salts of each of the foregoing compounds, wherein
R1 is selected from the group consisting of
xe2x80x94H,
xe2x80x94NO2,
xe2x80x94CN,
-halogen,
-lower alkyl which is straight-chained and which optionally may be substituted by the group consisting of xe2x80x94OH and halogen,
xe2x80x94OR5,
xe2x80x94R6OR7,
xe2x80x94COOR7,
xe2x80x94CONR8R9 (a.k.a. carboxamide),
xe2x80x94NR10R11,
xe2x80x94NHCOR12, and
xe2x80x94NHSO2R13;
R2 and R4 are each independently selected from the group consisting of
xe2x80x94H,
-halogen,
xe2x80x94NO2,
xe2x80x94CF3, and
-straight chained lower alkyl;
R3 is selected from the group consisting of
xe2x80x94H,
-lower alkyl which optionally may be substituted by xe2x80x94OH, xe2x80x94OR9, F, and aryl,
-cycloalkyl,
-aryl,
-heterocycle,
-heteroaryl,
xe2x80x94COOR7 
xe2x80x94CN,
-alkenyl,
xe2x80x94CONR8R9, and
-alkynyl;
R5 is selected from lower alkyl which optionally may be substituted by halogen;
R6 is selected from lower alkylene;
R7 is selected from the group consisting of xe2x80x94H and lower alkyl;
R8 and R9 are each independently selected from the group consisting of xe2x80x94H and -lower alkyl which itself optionally may be substituted by xe2x80x94OH and xe2x80x94NH2; alternatively, R8 and R9 may form a 5- or 6-membered heterocycle which optionally may be substituted by the group consisting of xe2x80x94OH, xe2x80x94NH2, and lower alkyl;
R10, R11 and R12 are each independently selected from the group consisting of xe2x80x94H and lower alkyl;
R13 is selected from the group consisting of lower alkyl which optionally may be substituted by the group consisting of halogen and xe2x80x94NR14R15; and
R14 and R15 are each independently selected from the group consisting of xe2x80x94H and lower alkyl which optionally may be substituted Halogen, or alternatively, xe2x80x94NR14R15 is a heterocycle.
The present invention is further directed to pharmaceutical compositions comprising a pharmaceutically effective amount of any one or more of the above-described compounds, or a pharmaceutically acceptable salt or prodrug thereof, and a pharmaceutically acceptable carrier or excipient.
The present invention is also directed to a method for treating solid tumors, in particular breast, colon, lung and prostate tumors, more specifically breast and colon tumors, by administering to a human patient in need of such therapy an effective amount of a compound of formula I, its salts or prodrugs.
As used herein, the following terms shall have the following definitions.
xe2x80x9cArylxe2x80x9d means an aromatic group having 5 to 10 atoms and consisting of 1 or 2 rings. Examples of aryl groups include phenyl and 1- or 2-naphthyl.
xe2x80x9cAlkenylxe2x80x9d means a straight-chain or branched, substituted or unsubstituted, aliphatic unsaturated hydrocarbon having 2 to 6, preferably 2 to 4, carbon atoms and containing double bonds. Typical alkenyl groups include ethylene, propylene, isopropylene, butylene and the like. Preferred alkenyl groups are straight-chained.
xe2x80x9cAlkynylxe2x80x9d means a straight-chain or branched, substituted or unsubstituted, aliphatic unsaturated hydrocarbon having 2 to 6, preferably 2 to 4, carbon atoms and containing triple bonds. Typical alkynyl groups include acetylene and the like. Preferred alkynyl groups are straight-chained.
xe2x80x9cCycloalkylxe2x80x9d means a non-aromatic, partially or completely saturated cyclic aliphatic hydrocarbon group containing 3 to 8 atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
xe2x80x9cEffective Amountxe2x80x9d means an amount of at least one compound of Formula I, or a pharmaceutically acceptable salt, prodrug or metabolite thereof, that significantly inhibits proliferation of a tumor cell, including human tumor cell lines.
xe2x80x9cHalogenxe2x80x9d means fluorine, chlorine, bromine or iodine. Preferred halogens are fluorine and chlorine.
xe2x80x9cHeteroarylxe2x80x9d groups are aromatic groups having 5 to 10 atoms, one or 2 rings, and containing one or more hetero atoms. Examples of heteroaryl groups are 2-, 3- or 4-pyridyl, tetrazolyl, oxadiazolyl, pyrazinyl, quinolyl, pyrrolyl, and imidazolyl.
xe2x80x9cHetero atomxe2x80x9d means an atom selected from N, O and S.
xe2x80x9cHeterocyclexe2x80x9d means a 3- to 10-membered non-aromatic, partially or completely saturated hydrocarbon group, such as tetrahydroquinolyl, which contains one or two rings and at least one hetero atom.
xe2x80x9cIC50xe2x80x9d refers to the concentration of a particular pyrazolobenzodiazepine required to inhibit 50% of a specific measured activity. IC50 can be measured, inter alia, as is described in Example 4, infra.
xe2x80x9cLower Alkylxe2x80x9d denotes a straight-chain or branched, substituted or unsubstituted, saturated aliphatic hydrocarbon having 1 to 6, preferably 1 to 4, carbon atoms. Typical lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, 2-butyl, pentyl, hexyl and the like. Analogously, xe2x80x9clower alkylenexe2x80x9d means an organic radical derived from lower alklyl, for example ethylene-, propylene-, and the like.
xe2x80x9cPharmaceutically acceptable saltxe2x80x9d refers to conventional acid-addition salts or base-addition salts which retain the biological effectiveness and properties of the compounds of formula I and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Sample acid-addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Sample base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethylammonium hydroxide.
xe2x80x9cPharmaceutically acceptable,xe2x80x9d such as pharmaceutically acceptable carrier, excipient, prodrug, etc., means pharmacologically acceptable and substantially non-toxic to the subject to which the particular compound is administered.
xe2x80x9cPharmaceutically active metabolitexe2x80x9d means a metabolic product of a compound of formula I which is pharmaceutically acceptable and effective.
xe2x80x9cProdrugxe2x80x9d refers to a compound that may be converted under physiological conditions or by solvolysis to any of the compounds of formula I or to a pharmaceutically acceptable salt of a compound of formula I. A prodrug may be inactive when administered to a subject but is converted in vivo to an active compound of formula I.
xe2x80x9cSubstituted,xe2x80x9d as in substituted alkyl, means that the substitution can occur at one or more positions and, unless otherwise indicated, that the substituents at each substitution site are independently selected from the specified options.
In one embodiment, the current invention is directed to compounds having the formula: 
and prodrugs and pharmaceutically active metabolites of compounds of formula I, and the pharmaceutically acceptable salts of the foregoing compounds, wherein R1 through R15 are as defined above.
In a preferred embodiment of the compounds of formula I, R1 is on the 7 or 8 position and is selected from the group consisting of xe2x80x94H, xe2x80x94NO2, xe2x80x94CN, Halogen and unsubstituted lower alkyl. Preferred lower alkyls are methyl and ethyl.
In another preferred embodiment of the compounds of formula I, R2 is on the 2xe2x80x2 position and is selected from the group consisting of xe2x80x94H and -Halogen.
In another preferred embodiment of the compounds of formula I, R3 is selected from the group consisting of unsubstituted lower alkyl, cycloalkyl, heterocycle, and heteroaryl. Preferred lower alkyl groups are methyl and ethyl. Preferred cycloalkyl groups are unsubstituted C3-C5.
In another preferred embodiment of the compounds of formula I, R4 is at the 4xe2x80x2 position and is selected from the group consisting of xe2x80x94H and -Halogen, most preferably R4 is H.
In another preferred embodiment of the compounds of formula I, R5 and R6 are independently selected from methyl or ethyl, each of which optionally may be substituted by halogen. More preferably, R5 is trifluoromethyl.
In another preferred embodiment of the compounds of formula I, R7 is selected from the group consisting of xe2x80x94H, methyl and ethyl.
In another preferred embodiment of the compounds of formula I, R7 and R8 are each independently selected from xe2x80x94H, methyl, ethyl and hydroxyethyl. When R8 and R9 form a heterocycle, preferred heterocycle groups are 6-membered, unsubstituted, groups that most preferably include two heteroatoms. Most preferred heteroatoms are selected from O and N.
In another preferred embodiment of the compounds of formula I, R10, R11, and R12 are each independently selected from the group consisting of xe2x80x94H, methyl and ethyl.
In another preferred embodiment of the compounds of formula I, R13 is lower alkyl which optionally may be substituted by halogen, most preferably R13 is methyl, ethyl, or trifluoromethyl.
In another preferred embodiment of the compounds of formula I, R14 and R15 are each independently selected from H, methyl, ethyl and heterocycle. Preferred heterocycles are 3-7membered rings that include at least one Nitrogen.
The following intermediates are also examples of additional preferred compounds according to the present invention: 
wherein R1, R2 and R4 are as defined above; 
wherein, in each of the immediately foregoing formulas, each of R1, R2, R3 and R4 are as previously defined herein. These intermediates are useful in the synthesis of compounds of formula I.
The compounds disclosed herein and covered by the above formulae may exhibit tautomerism or structural isomerism. It is intended that the invention encompasses any tautomeric or structural isomeric form of these compounds, or mixtures of such forms, and is not limited to any one tautomeric or structural isomeric form utilized within the formulae drawn above.
The compounds of the invention may be prepared by processes known in the art. Suitable processes for synthesizing these compounds are provided in the examples. Generally, these compounds may be prepared according to the synthesis schemes provided below. 
Compound 1 is either available from commercial sources or is synthesized by methods known in the art. 
wherein R1xe2x80x2 can be any of the options for R1 as defined above and, similarly, R3xe2x80x2 can be any of the options for R3 as defined above.
Several substitutions may be obtained by chemical modification of existing functional groups using known methods as is exemplified in scheme 4 above. For example, when the desired R1=NH2, this substitution may be obtained by reduction of the corresponding nitro group. Similarly, when the desired R1=NHRxe2x80x2 (where Rxe2x80x2=xe2x80x94COR12, xe2x80x94SO2R13, or xe2x80x94R10R11), this substitution may be obtained by reaction of the corresponding R1=NH2 compound with an acid halide or anhydride. When the desired R1=CONRRxe2x80x3 (where R=hydrogen or lower alkyl, and Rxe2x80x3=lower alkyl), this substitution may be obtained by reaction of the corresponding compound where R1=I, with carbon monoxide and a primary or secondary amine in the presence of a palladium catalyst.
In addition, if R3 in the starting material is CO2Et, standard chemical modification may be used to produce compounds having the following corresponding R3groups:
CH2OH (reduction); CHO (partial reduction); CH2NMe2 (reductive amination of the aldehyde); CH2OMe (alkylation of the alcohol); CHxe2x95x90CH2 (olefination of the aldehyde); CONRRxe2x80x3 (where R=H or lower alkyl and Rxe2x80x3=H or lower alkyl, aminolysis with the corresponding amine HNRRxe2x80x3 where R=H or lower alkyl and Rxe2x80x3=H or lower alkyl); CONHNHR (where R=H, lower alkyl or aryl) (hydrazinolysisxe2x80x94reaction with hydrazine); CN (dehydration of the amide CONH2).
In the foregoing schemes, compound 1 is either commercially available, for example from Sigma, or can be readily synthesized by methods known in the art. Thus, compound 2 is prepared from the corresponding lactam (compound 1) by the procedure of Sternbach et al., J. Org. Chem. 29:231 (1964) or by reaction with Lawesson""s reagent.
In an alternative embodiment, the present invention is directed to pharmaceutical compositions comprising at least one compound of formula I or a prodrug thereof, or a pharmaceutically acceptable salt of a compound of formula I or a prodrug of such compound.
These pharmaceutical compositions can be administered orally, for example, in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions or suspensions. They can also be administered rectally, for example, in the form of suppositories, or parenterally, for example, in the form of injection solutions.
The pharmaceutical compositions of the present invention comprising compounds of formula I, prodrugs of such compounds, or the salts thereof, may be manufactured in a manner that is known in the art, e.g. by means of conventional mixing, encapsulating, dissolving, granulating, emulsifying, entrapping, dragee-making, or lyophilizing processes. These pharmaceutical preparations can be formulated with therapeutically inert, inorganic or organic carriers. Lactose, corn starch or derivatives thereof, talc, steric acid or its salts can be used as such carriers for tablets, coated tablets, dragees and hard gelatin capsules. Suitable carriers for soft gelatin capsules include vegetable oils, waxes and fats. Depending on the nature of the active substance, no carriers are generally required in the case of soft gelatin capsules. Suitable carriers for the manufacture of solutions and syrups are water, polyols, saccharose, invert sugar and glucose. Suitable carriers for injection are water, alcohols, polyols, glycerine, vegetable oils, phospholipids and surfactants. Suitable carriers for suppositories are natural or hardened oils, waxes, fats and semi-liquid polyols.
The pharmaceutical preparations can also contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifying agents, sweetening agents, coloring agents, flavoring agents, salts for varying the osmotic pressure, buffers, coating agents or antioxidants. They can also contain other therapeutically valuable substances, including additional active ingredients other than those of formula I.
As mentioned above, the compounds of formula I, prodrugs thereof, and their salts, and compositions containing these compounds are useful in the treatment or control of cell proliferative disorders, in particular oncological disorders. These compounds and formulations containing said compounds are particularly useful in the treatment or control of solid tumors, such as, for example, breast and colon tumors.
A therapeutically effective amount of a compound in accordance with this invention means an amount of compound that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the skill in the art.
The therapeutically effective amount or dosage of a compound of formula I can vary within wide limits and will be adjusted to the individual requirements in each particular case. In general, in the case of oral or parenteral administration to adult humans weighing approximately 70 Kg, a daily dosage of about 10 mg to about 10,000 mg, preferably from about 200 mg to about 1,000 mg, should be appropriate, although the upper limit may be exceeded when indicated. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, it may be given as continuous infusion.